The experiments described in this proposal seek to establish whether infection of humans with M. tuberculosis and/or HIV-1 result in perturbations of intracellular signalling pathways leading to macrophage activation and NK- and T-cell differentiation. Two major insights will derive from this line of investigation. First, we will determine whether natural killer cells and T cells remain responsive to IL-2, and whether monocytes and macrophages retain the capacity to be activated by interferon-gamma, during the progression of disease in patients infected with M. tuberculosis and HIV-1. Second, constitutive activation of these pathways in cells isolated from patients, and from patients undergoing cytokine therapy, will serve as an index of the production of these cytokines in vivo. IL-2-induced signaling in NK- and T-cells will be assessed by measuring protein tyrosine phosphorylation, the activation of the protein tyrosine kinase p56lck, the activation of protein kinase C (PKC), the activation of serine/threonine kinase Raf-1 and the induction of IL-2-specific immediate early genes. IFNgamma responsiveness in macrophages and monocytes will be assessed by determining the levels IFNgamma induced gene products including the 48K myristoylated PKC substrate, IP-10 and the high affinity Fcgamma-receptor. We will also examine whether cell wall products of M. tuberculosis such as MDP and LAM B prime monocytes for enhanced PKC-dependent responses such as those leading to increased arachidonic acid metabolism. It will be determined whether priming correlates with the expression and phosphorylation of the MARCKS and 42K PKC substrates. We will also determine whether M. tuberculosis and its cell wall products activate the NF-kB transcriptional pathway, and whether this results in the activation of latent HIV-1. Finally, we will identify and characterize a plasma membrane receptor for the Pr55gag protein of HIV-1 in T-cells and macrophages. To this end, the precise membrane binding domain of Pr55gag will be defined by deletional mutagenesis and a synthetic peptide, based on this domain, will be used in crosslinking experiments to identify the membrane receptor to which Pr55gag binds. The identification of such a receptor will provide new insights into the biology of HIV-1 and identify a potential target for rational drug design which could lead to successful therapeutic intervention.